A major cause of morbidity and mortality in the aged is infections, including common viral infections such as influenza and respiratory syncytial virus (RSV), that the immune system of healthy young individuals has little difficulty combating. While there are likely to be multiple causes of age-related immune decline, a key problem is thymic involution, which results in decreased production of new T cells (recent thymic emigrants, RTE). Work by ourselves and others has demonstrated that in contrast to the young thymus, aging is marked by thymic involution due to the loss of thymic epithelial cells (TEC), which produce the signals needed in the thymus for progenitor cell expansion and differentiation, notably IL-7 and Kit ligand (KL). Using both chronologically aged mice and the Klotho premature aging models, we have shown that thymic involution can be reversed by cytokine-mediated (keratinocyte growth factor) expansion and maintenance of TEC, resulting in normalization of T-cell production, peripheral T lymphocyte numbers and of immune responsiveness. The effects of KGF are transient, lasting approximately 3 weeks, but repeated dosing can maintain the restored thymic microarchitecture and T-cell production. The transient effects suggest that KGF-responsive TEC progenitors continue to rapidly senesce. An alternative approach to restoring thymic function in aging is to transplant stem-cell derived TEC progenitors into aged mice. During normal embryogenesis, TEC develop from endodermal progenitors. The DiGeorge syndrome (DGS) is a common pediatric malformation complex that includes immune deficiency due to developmental failure of TEC. The T- box gene Tbx1 is located in the DGS critical region, and targeted mutation of Tbx1 results in a DGS phenotype in mice, including athymia. We have been able to derive functional TEC from murine embryonic stem cells (mESC). Definitive endoderm was generated by Activin A stimulation of mESC, and the cells were then lentivirally transduced with the Tbx1 gene. The definitive endoderm-Tbx1 cells (DE-Tbx1) were then co-cultured with embryonic thymic mesenchymal cells. The resultant cells express several markers of TEC, and have the ability to induce T-cell differentiation in vitro in thymic re-aggregation assays. In the proposed studies, the ability of the mESC-derived TEC-like cells will be tested in the Klotho premature aging mouse model to determine whether they can engraft and support in vivo thymopoiesis. We hypothesize that 1) mESC-derived TEC will recapitulate normal TEC ontogeny;2) that mESC derived TEC progenitors can engraft in the Klotho murine model of premature aging and correct thymic involution;3) mESC-derived TEC will maintain their ontological age, e.g., their gene expression signature will be that of "young" TEC, rather than aged TEC. The studies will provide essential information regarding the generation, transplantability and efficacy of mESC-derived TEC in the correction of age-related thymic involution. PUBLIC HEALTH RELEVANCE: T lymphocytes are cells of the immune system that control the body's response to infection. The thymus makes T lymphocytes in early life, but production of T lymphocytes profoundly decreases during aging. The studies will examine how embryonic stem cells can be used to generate thymic epithelial cells, which can be transplanted into mice that undergo premature thymic aging to regenerate the thymus and restore the ability to make T lymphocytes.